Oxidized Nicotinamide Adenine Dinucleotide Research Articles

Theoretical and Experimental Study of the Homogeneous Catalytic Oxidation of Nicotinamide Adenine Dinucleotide (NADH) at Spherical Gold Electrodes Using Linear Sweep Voltammetry and Chronopotentiometry

AbstractThe homogeneous catalytic oxidation of coenzyme nicotinamide adenine dinucleotide (NADH) with dopamine at a thiolactic acid modified gold spherical electrode of radius 0.0128 cm has been studied in aqueous phosphate media (pH 7.0) using linear sweep voltammetry and chronopotentiometry with power time and exponential time currents. We have proved that, under the conditions of this study, the steady state is reached, in such a way that the expressions for the I−E and dI/dE−E responses are simplified and become independent of time and the electrochemical technique employed. Therefore, the experimental I−E and dI/dE−E curves obtained in chronopotentiometry and linear sweep voltammetry are practically superimposable and, from easy expressions for the peak parameters of the dI/dE−E curves deduced in this paper, values of the chemical and electrochemical rate constants have been obtained.

Age‐related decreases in NAD(P)H and glutathione cause redox declines before ATP loss during glutamate treatment of hippocampal neurons

Age-related glutamate excitotoxicity depends in an unknown manner on active mitochondria, which are key determinants of the cellular redox potential. Compared with embryonic and middle-aged neurons, old-aged rat hippocampal neurons have a lower resting reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and a lower redox ratio (NAD(P)H/flavin adenine nucleotide). Glutamate treatment resulted in an initial increase in NAD(P)H concentrations in all ages, followed by a profound calcium-dependent, age-related decline in NAD(P)H concentration and redox ratio. With complex I of the electron transport chain inhibited by rotenone, treatment with glutamate or ionomycin only resulted in the increase in NAD(P)H fluorescence. High-performance liquid chromatography analysis of adenine nucleotides in brain extracts showed 50% less nicotinamide adenine dinucleotide (NADH) and almost twice as much oxidized nicotinamide adenine dinucleotide, demonstrating a more oxidized ratio in old than middle-aged brain. Resting glutathione content also declined with age and further decreased with glutamate treatment without accompanying changes in adenosine triphosphate levels. We conclude that age does not affect production of NADH by dehydrogenases but that old-aged neurons consume more NADH and glutathione, leading to a catastrophic decline in redox ratio.

The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis

Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival.

Electrocatalytic activity of 2,3,5,6-tetrachloro-1,4-benzoquinone/multi-walled carbon nanotubes immobilized on edge plane pyrolytic graphite electrode for NADH oxidation

This work reports the electrocatalytic activity of 2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ)/multi-walled carbon nanotubes (MWCNT) immobilized on an edge plane pyrolytic graphite electrode for nicotinamide adenine dinucleotide (NADH) oxidation. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS) were used to confirms the presence of chloro after the nanotube modification with 2,3,5,6-tetrachloro-1,4-benzoquinone. The surface charge transfer constant, k s, and the charge transfer coefficient for the modified electrode, α, were estimated as 98.5 (±0.6) s −1 and 0.5, respectively. With this modified electrode the oxidation potential of the NADH was shifted about 300 mV toward a less positive value, presenting a peak current much higher than those measured on an unmodified edge plane pyrolytic graphite electrode (EPPG). Cyclic voltammetry and rotating disk electrode (RDE) experiments indicated that the NADH oxidation reaction involves 2 electrons and a heterogenous rate constant ( k obs) of 3.1 × 10 5 mol −1 l s −1. The detection limit, repeatability, long-term stability, time of response and linear response range were also investigated.

Production of l-alanine by metabolically engineered Escherichia coli

Escherichia coli W was genetically engineered to produce L: -alanine as the primary fermentation product from sugars by replacing the native D: -lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native D: -lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of L: -alanine to D: -alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l(-1) glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g(-1) glucose) with a chiral purity greater than 99.5% L: -alanine.

A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD+), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10−4–4.5×10−3 M, determination limit was 3.9×10−4 M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD+ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.

TEMPORAL WINDOW OF METABOLIC BRAIN VULNERABILITY TO CONCUSSIONS

In the present study, we investigate the existence of a temporal window of brain vulnerability in rats undergoing repeat mild traumatic brain injury (mTBI) delivered at increasing time intervals. Rats were subjected to two diffuse mTBIs (450 g/1 m height) with the second mTBI delivered after 1 (n = 6), 2 (n = 6), 3 (n = 6), 4 (n = 6), and 5 days (n = 6) and sacrificed 48 hours after the last impact. Sham-operated animals were used as controls (n = 6). Two further groups of six rats each received a second mTBI after 3 days and were sacrificed at 120 and 168 hours postinjury. Concentrations of adenine nucleotides, N-acetylated amino acids, oxypurines, nucleosides, free coenzyme A, acetyl CoA, and oxidized and reduced nicotinamide adenine dinucleotides, oxidized nicotinamide adenine dinucleotide phosphate, and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate nicotinic coenzymes were measured in deproteinized cerebral tissue extracts (three right and three left hemispheres), whereas the gene expression of N-acetylaspartate acylase, the enzyme responsible for N-acetylaspartate (NAA) degradation, was evaluated in extracts of three left and three right hemispheres. A decrease of adenosine triphosphate, adenosine triphosphate/adenosine diphosphate ratio, NAA, N-acetylaspartylglutamate, oxidized and reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, and acetyl CoA and increase of N-acetylaspartate acylase expression were related to the interval between impacts with maximal changes recorded when mTBIs were spaced by 3 days. In these animals, protracting the time of sacrifice after the second mTBI up to 1 week failed to show cerebral metabolic recovery, indicating that this type of damage is difficult to reverse. A metabolic pattern similar to controls was observed only in animals receiving mTBIs 5 days apart. This study shows the existence of a temporal window of brain vulnerability after mTBI. A second concussive event falling within this time range had profound consequences on mitochondrial-related metabolism. Furthermore, because NAA recovery coincided with normalization of all other metabolites, it is conceivable to hypothesize that NAA measurement by 1H-NMR spectroscopy might be a valid tool in assessing full cerebral metabolic recovery in the clinical setting and with particular reference to sports medicine in establishing when to return mTBI-affected athletes to play. This study also shows, for the first time, the influence of TBI on acetyl-CoA, N-acetylaspartate acylase gene expression, and N-acetylaspartylglutamate, thus providing novel data on cerebral biochemical changes occurring in head injury.

Acute, Severe Hypoglycemia Occurring During General Anesthesia in a Nondiabetic Adult

To the Editor: Hypoglycemia during general anesthesia is rarely reported in the nonpediatric, nondiabetic population. Here we report the unexpected development of intraoperative hypoglycemia in a 26-year-old Uzbekhastani male management consultant who presented emergently for maxillofacial surgery. He had no medical problems, allergies, significant family history, or previous problems during general anesthesia. Preoperative laboratory studies including liver function tests were normal. He was not intoxicated on admission and had not taken any other prohibited substances. A routine blood glucose level of 84 mg/dL had been recorded on the same morning of hospital admission. Because of other operating room emergencies, the patient arrived in the operating room having been NPO for 14 h; he last ate at midnight after the consumption of approximately two bottles of wine accompanying a meal during the course of the same evening. Five minutes after IV induction [fentanyl (100 μg), propofol (200 mg), and vecuronium (8 mg)] and maintenance of anesthesia (sevoflurane/air/oxygen:MAC 1.3), marked diaphoresis suddenly appeared. No change in blood pressure or heart rate was noted. No skin pallor or neurological, including pupillary abnormalities were noted. Both ETCO2 and esophageal temperature were within normal limits. Blood glucose was 35 mg/dL (Bayer Ascensia Elite glucometer, Bayer AG, Leverkusen, Germany). Exactly 30 mL of 50% glucose was administered IV where upon the profuse diaphoresis stopped. Blood glucose returned to, and remained within, normal limits thereafter. Emergence from anesthesia, extubation, and postoperative recovery were unremarkable. The patient had no neurological sequelae and was discharged home the next day. Alcoholic hypoglycemia occurs in people who ingest alcohol after fasting long enough to make hepatic glucose production dependent on gluconeogenesis (1). In the absence of both gluconeogenesis and intestinal absorption of glucose, the liver contains enough glycogen to maintain normoglycemia for 8–10 hours. Hepatic alcohol oxidation increases the cytosolic ratio of reduced to oxidized nicotinamide adenine dinucleotide (NAD). Consequently, this reversed NAD ratio inhibits hepatic glucose release by inhibiting major plasma gluconeogenic substrates (lactate, alanine) for glucose synthesis. The incidence of ethanol-induced profound hypoglycemia is very low (2), with approximately 1% of patients with ethanol levels greater than 0.10% sustaining severe hypoglycemia (serum glucose <50 mg/dL). Recent studies confirm that cognitive dysfunction is the most sensitive marker of severe hypoglycemia (3). However, blood glucose levels may be close to, or within, the hypoglycemic range but not necessarily severe enough to elicit clinically obvious cognitive dysfunction (2,3). Because anesthesia masks cognitive dysfunction, the most sensitive clinical marker of hypoglycemia, anesthesiologists should have a low threshold for measuring blood glucose levels in starved patients with a history of significant alcohol intake. Gareth L. Ackland, PhD, FRCA Centre for Anaesthesia [email protected] Megan Smith, MBBS Alan P. McGlennan, FRCA Department of Anesthesia University College Hospital London, UK

Fabrication and characterization of Meldola's blue/zinc oxide hybrid electrodes for efficient detection of the reduced form of nicotinamide adenine dinucleotide at low potential

We report the synthesis and the electrochemical properties of hybrid films made of zinc oxide (ZnO) and Meldola's blue dye (MB) using cyclic voltammetry (CV). MB/ZnO hybrid films were electrochemically deposited onto glassy carbon, gold and indium tin oxide-coated glass (ITO) electrodes at room temperature (25 ± 2 °C) from the bath solution containing 0.1 M Zn(NO 3) 2, 0.1 M KNO 3 and 1 × 10 −4 M MB. The surface morphology and deposition kinetics of MB/ZnO hybrid films were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical quartz crystal microbalance (EQCM) techniques, respectively. SEM and AFM images of MB/ZnO hybrid films have revealed that the surfaces are well crystallized, porous and micro structured. MB molecules were immobilized and strongly fixed in a transparent inorganic matrix. MB/ZnO hybrid films modified glassy carbon electrode (MB/ZnO/GC) showed one reversible redox couple centered at formal potential ( E 0′) −0.12 V (pH 6.9). The surface coverage ( Γ) of the MB immobilized on ZnO/GC was about 9.86 × 10 −12 mol cm −2 and the electron transfer rate constant (ks) was determined to be 38.9 s −1. The MB/ZnO/GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the oxidation of nicotinamide adenine dinucleotide (NADH). The linear response range between 50 and 300 μM NADH concentration at pH 6.9 was observed with a detection limit of 10 μM (S/N = 3). The electrode was stable during the time it was used for the full study (about 1 month) without a notable decrease in current. Indeed, dopamine (DA), ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) did not show any interference during the detection of NADH at this modified electrode.

Electrocatalytic oxidation of NADH with Meldola's blue functionalized carbon nanotubes electrodes

Meldola's blue (MB) functionalized carbon nanotubes (CNT) nanocomposite film (MB/CNT) electrode was prepared by non-covalent adsorbing MB on the surface of a carbon nanotubes modified glassy carbon electrode (CNT/GCE). Electrochemical behaviors of the resulting electrode were investigated thoroughly with cyclic voltammetry in the potential range of −0.6 to 0.2 V, and two well-defined redox couples were clearly visualized. We also studied the electron transfer kinetics of MB loaded on CNT (MB/CNT) in comparison with that of MB on conventional graphite powder (MB/GP). The heterogeneous electron transfer rate constant ( k s) of MB/CNT was calculated to be about three times larger than that of MB/GP. The accelerated electron transfer kinetics was attributed to the unique electrical and nanostructural properties of CNT supports as well as the interaction between MB and CNT. In connection with the oxidation of nicotinamide adenine dinucleotide (NADH), excellent electrocatalytic activities were observed at MB/CNT/GCE compared with MB/GP modified glassy carbon electrode (MB/GP/GCE). Based on the results, a new NADH sensor was successfully established using the MB/CNT/GCE. Under a lower operation potential of −0.1 V, NADH could be detected linearly up to a concentration of 500 μM with an extremely lower detection limit of 0.048 ± 0.02 μM estimated at a signal-to-noise ratio of 3. Sensitivity, selectivity, reproducibility and stability of the NADH sensor were also investigated and the main analytical data were also compared with those obtained with the MB/GP/GCE.

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD +) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP +), formed following the reaction of NADK with NAD + and adenosine 5′-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP +–isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.

Electrochemical properties and electrocatalytic activity of hematoxylin modified carbon paste electrode toward the oxidation of reduced nicotinamide adenine dinucleotide (NADH)

The electrocatalytic oxidation of nicotinamide adenine dinucleotide (NADH) was studied on a hematoxylin modified carbon paste electrode (HMCPE), using cyclic voltammetry, chronoamperometry and differential pulse voltammetry as diagnostic techniques. The surface charge transfer rate constant, k s, and the charge transfer coefficient, α c, for electron transfer between the CPE and the spiked hematoxylin were estimated as 89.56 ± 0.75 s −1 and 0.52, respectively, in pH 7.0, according to the procedure of Laviron. The cyclic voltammograms of the modified electrode indicated that the reactivity of spiked hematoxylin was strongly dependent on the solution pH. Two linear segments were found with slope values of −59.0 and −28.3 mV/pH in the ranges of pH 2.0–8.0 and 8.0–10.0, respectively. The hematoxylin modified electrode showed excellent electrocatalytic activity toward NADH oxidation in a phosphate buffer solution (pH 7.0) with a diminution of the overpotential of about 280 mV, compared to the process at an unmodified electrode. The electron transfer coefficient, α c, and the heterogeneous rate constant, k h, for oxidation of NADH at the hematoxylin modified electrode were also determined as 0.22 and 2.15 × 10 −3 cm s −1, respectively, by cyclic voltammetry measurements. Differential pulse voltammetry (DPV) exhibited a wide linear dynamic ranges and a lower detection limit of 0.08 μM for NADH.

A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of α-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340 nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca 2+ concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

Electrochemical evaluation of coumestan modified carbon paste electrode: Study on its application as a NADH biosensor in presence of uric acid

The electrocatalytic oxidation of nicotinamide adenine dinucleotide (NADH) has been studied on a coumestan derivative modified carbon paste electrode (CMCPE), using cyclic voltammetry, chronoamperometry and differential pulse voltammetry as diagnostic techniques. The surface charge transfer rate constant, k s, and the charge transfer coefficient, α, for electron transfer between the CPE and the spiked derivative coumestan were estimated in different pHs, according to the procedure of Laviron. The cyclic voltammograms of the modified electrode indicate that the reactivity of spiked derivative coumestan is strongly dependent on the solution pH. Two linear segments were found with slope values of −55.6 and −28.8 mV/pH in the ranges of pH 2.0–9.7 and 9.7–12.0, respectively. The CMCPE shows excellent electrocatalytic activity toward NADH oxidation in phosphate buffer solution (pH 7.0) with a diminution of the overpotential of about 240 mV compared to the process at an unmodified electrode. The heterogeneous rate constant for oxidation of NADH at the CMCPE was also determined by cyclic voltammetry and chronoamperometry measurements. Differential pulse voltammetry (DPV) exhibits two linear dynamic ranges and a lower detection limit of 0.10 μM for NADH. Finally, the CMCPE could separate the oxidation peak potentials of NADH and uric acid present in the same solution, though at the unmodified CPE the peak potentials were indistinguishable.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Electrocatalytic oxidation of NADH at single-wall carbon-nanotube-paste electrodes: kinetic considerations for use of a redox mediator in solution and dissolved in the paste

Cyclic voltammetry has been successfully used to study the oxidation of nicotinamide adenine dinucleotide (NADH) at single-wall carbon-nanotube-paste (CNTP) electrodes modified with p-methylaminophenolsulfate (p-MAP) and 3,4-dihydroxybenzaldehyde (3,4-DHB). Diffusion-like behaviour was observed for p-MAP-modified electrodes, and a diffusion coefficient of 2.4x10(-6) cm2 s(-1) was calculated for p-MAP in the paste. The behaviour of 3,4-DHB-modified CNTP electrodes was typical of that of surface-confined mediators. p-MAP electrocatalytic activity was first checked in solution, and a rate constant of 9.2 mol(-1) L s(-1) was obtained for the reaction between NADH and the mediator. The p-MAP-modified electrode did not have significant electrocatalytic activity for electro-oxidation of NADH, probably because of the formation of a complex between NADH and the confined mediator. In contrast, the 3,4-DHB-modified electrode had very good NADH electrocatalytic activity, with a heterogeneous rate constant of approximately 20x10(2) mol(-1) L s(-1).

Characterization of oxidized nicotinamide adenine dinucleotide (NAD +) analogues using a high-pressure-liquid-chromatography-based NAD +-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD +)-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD +) and products (adenosine 5′-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD + analogue, 2′-F-ribo-NAD +, was a competing substrate or a competitive inhibitor for this toxin. This NAD + analogue was hydrolyzed at a rate of 0.2% that of NAD + yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD + analogue, ε-NAD +, is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD + as determined using HPLC. The rate of hydrolysis of ε-NAD + calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD +-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD + and its analogues.

Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay

While many investigations measuring oxidized nicotinamide adenine dinucleotide (NAD +) and reduced nicotinamide adenine dinucleotide (NADH) have been carried out on several mammalian tissues and blood cells, few reports have dealt with monolayers of cultured cells. Here we show a novel method to measure NAD + and NADH in monolayers of a neuroblastoma cell line. The method was established by modifying a single extraction procedure originally developed for erythrocytes and an enzymatic cycling assay using a dye that absorbs in visible range. The following modifications were made. (i) Addition of 0.05% of a detergent, Triton X-100, to carbonate–bicarbonate extraction buffer enabled us to accurately measure cellular [NADH]/([NAD +] + [NADH]). (ii) Addition of N-ethyldibenzopyrazine ethyl sulfate salt (phenazine ethosulfate) immediately before the incubation suppressed the gradual decline of the sensitivity of the assay. The procedure presented here provides a simple and inexpensive measurement of NAD + and NADH in cell monolayers.

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP + Sia → CMP-Sia + pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia → pyruvate + ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate + NADH → lactate + oxidized nicotinamide adenine dinucleotide (NAD +), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.

A Comparative Proteomic Strategy for Subcellular Proteome Research

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.